Glucagon-like peptide-2 (GLP-2) is a 33 amino acid intestinotrophic peptide hormone generated via post-translational processing of proglucagon (Orskov et al., FEBS Lett. 247: 193-196 (1989); Hartmann et al., Peptides 21: 73-80 (2000)). In mammals, GLP-2 is liberated from proglucagon in the intestine and brain but not in pancreas, as a result of cell-specific expression of prohormone convertases in gut endocrine cells (Dhanvantari et al., Mol. Endocrinol. 10: 342-355 (1996); Rothenberg et al., Mol. Endocrinol. 10: 334-341 (1996); Damholt et al., Endocrinology 140: 4800-4808 (1999); Hoist, Trends Endocrinol Metab. 10: 229-235 (1999)). Analysis of rat and human plasma using a combination of high-performance liquid chromatography and site-specific GLP-2 antisera reveals the presence of two principal circulating molecular forms, GLP-21-33 and GLP-233-33 (Hartmann et al., Supra; Brubaker et al., Endocrinol. 138: 4837-4843 (1997); Hartmann et al., J. Clin. Endocrinol. Metab. 85: 2884-2888 (2000)). GLP-21-33 is cleaved in vivo by the protease dipeptidyl peptidase IV (DPP IV), which removes the first two residues, histidine and alanine (HA). The resulting peptide GLP-23-33 is essentially inactive.
GLP-2 regulates gastric motility, gastric acid secretion, intestinal hexose transport, and increases the barrier function of the gut epithelium (reviewed in Drucker, J. Clin. Endocr. Metab. 86: 1759-1764 (2001)). It significantly enhances the surface area of the mucosal epithelium via stimulation of crypt cell proliferation and inhibition of apoptosis in the enterocyte and crypt compartments. (Drucker et al., Proc. Natl. Acad. Sci. U.S.A. 93: 1911-7916 (1996)). GLP-2 reduces mortality and decreases mucosal injury, cytokine expression, and bacterial septicemia in small and large bowel inflammation (Boushey et al., Am. J. Physiol. 277: E937-E947 (1999); Prasad et al., J. Pediatr. Surg. 35: 357-359 (2000)). GLP-2 also enhances nutrient absorption and gut adaptation in rodents or humans with short bowel syndrome (SBS) (Jeppesen et al., Gastroenterology 120: 806-815 (2001)).
The actions of GLP-2 are transduced by the GLP-2 receptor (GLP-2R), a G protein-coupled receptor expressed in gut endocrine cells of the stomach, small bowel, colon, as well as enteric neurons and subendothelial myofibroblasts (Munroe et al., Proc. Natl. Acad. Sci U.S.A. 96: 1569-1573 (1999); Yusta et al., Gastroenterology 119: 744-755 (2000); Bjerknes et al., Proc. Natl. Acad. Sci. U.S.A. 98: 12497-12502 (2001); Orskov et al., Regul. Pept. 124: 105-112 (2005)). Direct activation of GLP-2R signaling in transfected baby hamster kidney fibroblasts expressing the GLP-2 receptor (BHK-GLP-2R cells) confers resistance to cycloheximide-induced apoptosis (Yusta et al., J. Biol. Chem. 275: 35345-35352 (2000)).
The cytoprotective, reparative, and energy-retentive properties of GLP-2 suggest that GLP-2 may potentially be useful for the treatment of human disorders characterized by injury and/or dysfunction of the intestinal mucosal epithelium. Intestinal epithelial injury is seen in patients with inflammatory bowel disease (IBD), including Crohn's Disease and ulcerative colitis, and in patients with autoimmune diseases that are associated with an inflammatory response in the intestine, such as Celiac's Disease (reviewed in Hanauer, New England J. Med. 334: 841-848 (1996)). In addition, some chemotherapy drugs cause injury to the intestinal epithelium that result in toxic side effects that are dose limiting (Oster, Oncology 13: 41 (1999)). Increased intestinal permeability is also reported in cases of acute pancreatitis (Kouris et al., Am. J. Surg. 181: 571-575 (2001)) and could contribute to food allergies by allowing macromolecules to access the subendothelial compartment (Troncone et al., Allergy 49: 142-146 (1994)).
As an important regulatory hormone in nutrient absorption, GLP-2 is also promising in treating patients with short bowel syndrome (SBS) (Drucker et al., Supra; Rubin, Gastroenterol. 117:261-263 (1999); Nightingale, Gut 45: 478-479 (1999)). SBS is defined as malabsorption resulting from anatomical or functional loss of a significant length of the small intestine (reviewed in Jeppesen, J. Nutr. 133: 3721-3724 (2003)). The causes of short bowel syndrome differ between adults and children: in adults, it most often results after surgery for Crohn's disease or mesenteric infarction; while in infants, the causes more commonly include necrotizing enterocolitis, gastroschisis, atresia, and volvulus (Platell et al., World J. Gastroenterol. 8: 13-20 (2002)).
Teduglutide, a DPP-IV resistant GLP-2 peptide analog (where alanine-2 is substituted with glycine (A2G)), is being developed for the potential treatment of gastrointestinal (GI) diseases, including SBS, Crohn's disease and pediatric GI disorders. Teduglutide also has potential for the treatment of mucositis associated with cancer chemotherapy and IBD. However, due to the peptide's low molecular weight, teduglutide is cleared quickly with a half-life of less than 30 minutes. Accordingly, daily dosing is required to maintain the therapeutic level (Shin et al., Curr. Opin. Endocrin. Diabetes 12: 63-71 (2005)). Therefore, a need exists for a modified GLP-2 that will overcome the short half-life while retaining its function and provide for facile development and manufacture.
Inflammatory ileus, the temporary impairment of coordinated gastrointestinal motility following invasive surgery or traumatic injury, remains a major clinical problem, extending hospital stays and often contributing to medical complications during the recovery period (Holte and Kehlet, Br. J. Surg. 87: 1480-1493 (2000)). Ileus is characterized by delayed gastric emptying, dilatation of the small bowel and colon, abdominal distension, loss of normal propulsive contractile patterns, and inability to evacuate gas or stool, leading to prolonged patient discomfort (abdominal distension, nausea, emesis).
In susceptible individuals, such as the elderly or patients with cardiopulmonary compromise, ileus can lead to more serious complications including acute gastric dilatation, cardiac arrhythmia, respiratory distress, aspiration pneumonia, and failure of surgical anastomoses. In severe cases, prolonged loss of the normal “housekeeping” contractile activity of the GI tract can contribute to bacterial overgrowth and breakdown of intestinal barrier function, followed by bacterial translocation and entry into the systemic circulation (Anup and Balasubramanian, J. Surg. Res. 92: 291-300 (2000)). This in turn can lead to endotoxemia, sepsis, multi-organ failure and ultimately death, an outcome for which elderly patients are the most susceptible. Even in the absence of complications, the return of normal bowel function is a prime limiting factor for release of patients from hospital, with inflammatory ileus increasing hospital stays by 3 to 5 days. Thus, costs accrued from increased morbidity and protracted hospital stays can be substantial.
Factors that contribute to the development and maintenance of ileus include the activation of central sympathetic inhibitory reflexes which release norepinephrine into the bowel wall, inhibitory humoral agents, anesthetic and analgesic agents, and inflammatory mediators (Livingston and Passaro, Dig. Dis. Sci. 35: 121-132 (1990); Bauer et al., Curr. Opin. Crit. Care 8: 152-157 (2002)). Results from rodent studies suggest that inflammation within the wall of the GI tract plays a central role in initiating and maintaining ileus.
Studies employing rodent models of post-operative ileus demonstrate that the muscularis externa is a highly immunologically active compartment. Normally resident within the muscularis externa is an impressive array of common leukocytes (Mikkelsen, Histol. Histopathol. 10: 719-736 (1995); Kalff et al., Ann. Surg. 228: 652-663 (1998)). Most abundant of these are resident macrophages, which form an extensive network of cells from the esophagus to the colon, and which are poised to defend the gastrointestinal tract from potential injury and disease. Disturbances to the bowel during abdominal surgery activate this macrophage network, initiating a local molecular inflammatory response. The activated macrophages release pro-inflammatory cytokines (IL-6, IL-1β, TNFα) and chemokines (MCP-1) that suppress neuromuscular communication within the muscularis and induce the expression of adhesion molecules (ICAM-1, P-selectin) on the vascular endothelium (Kalff et al., J. Leukoc. Biol. 63: 683-691 (1998); Josephs et al., J. Surg. Res. 86: 50-54 (1999); Kalff et al., Gastroenterology 117: 378-387 (1999); Kalff et al., Gastroenterology 118: 316-327 (2000); Wehner et al., Surgery 137: 436-46 (2005)). This in turn leads to a cellular inflammatory response characterized by recruitment of leukocytes (monocytes, neutrophils, T-cells, mast cells) from the systemic circulation, where there is a positive correlation between the magnitude of the inflammatory cell infiltrate and the severity of ileus (Kalff et al., Surgery 126: 498-509 (1999)). Infiltrating leukocytes release additional cytokines as well as prostaglandins, nitric oxide, proteases and reactive oxygen species that further contribute to neuromuscular dysfunction (von Ritter et al., Gastroenterology 97: 605-609 (1989); Bielefeldt and Conklin, Dig. Dis. Sci. 42: 878-884 (1997)).
To date, there are few options available in the clinic for management of inflammatory ileus. Prokinetics such as cisapride and neostigmine have been shown to improve postoperative bowel motility (Shibata and Toyoda, Surg. Today 28: 787-791 (1998)). However, results are inconsistent and these drugs have an increased risk of adverse cardiovascular effects that have proven difficult to predict in terms of severity and patient susceptibility. COX-2 inhibitors have been shown in animal studies to be protective against postoperative dysmotility of the small bowel (Schwarz et al., Gastroenterology 121: 1354-1371 (2001)), but had little effect on colonic dysmotility (Turler et al., Anal. Surg., 231(1): 56-66 (2002)). Phase I clinical trials in humans were completed comparing celecoxib and rofecoxib (Bouras et al., Neurogastroenterol. Motil. 16: 729-735 (2004)), and neither agent was found to improve post-operative motility.
The rapid return to oral feeding after surgery has been promoted as a means to stimulate normal hormonal regulation of motility patterns. This was found to hasten the return of bowel function and to improve comfort in a subset of patients when used as part of a multi-modal approach to bowel rehabilitation (Holte & Kehlet, Minerva. Anestesiol., 68(4): 152-156 (2002)). However, this treatment did not result in a significant reduction in the length of hospital stay. Furthermore, adequate stimulation of hormonal patterns requires a threshold caloric load that many patients are unable to tolerate.
One of the most common factors contributing to the development of prolonged ileus is the administration of opioid analgesics for postoperative pain relief. Opioids exert their analgesic effects by interacting with one or more of three receptor subtypes present on neurons in the pain processing centers of the brain. Most current opioid analgesics, such as morphine, work primarily by activating μ-(mu) and δ (delta)-opioid receptors. However, these same receptors are also expressed on the neurons within the gastrointestinal tract that control bowel motility. Activation of the receptors, whether in the presence or absence of inflammatory ileus, significantly suppresses gastrointestinal contractile function, causing bowel stasis and constipation. Adalor Corporation has conducted Phase I and II clinical trials using Alvimopan, a peripherally restricted and selective μ-OR antagonist that does not cross the blood-brain barrier. When given in conjunction with opioid analgesics, Alvimopan prevented opioid-induced suppression of intestinal motility (Gonenne et al., Clin. Gastroenterol. Hepatol. 3: 784-791 (2005)). When compared with placebo, Alvimopan was found to hasten return of bowel function and to shorten hospital stay in patients who were experiencing mild to moderate postoperative ileus after having undergone abdominal surgery (Viscusi et al., Surg. Endosc. 20: 64-70 (2006)). Alvimopan does not alter inflammation.
To date, there are no safe and reliable treatment options available for the treatment of inflammatory ileus. The most effective remedies currently available are supportive in nature or ameliorate the compounding effects of opioid analgesia. They do not address inflammation as the underlying cause of ileus. Therefore, a significant unmet medical need remains.